A Comparison Between the Effects of Estrogen and Soy Extract on Chronic Pain in Male Rats

In addition to its effect on reproductive organs, estrogen exhibits complex effects on brain structure, function and behavior. In this regard, the influence of gonadal hormones especially estrogens on nociception has recently been accepted. Soybean is known as an important phytoestrogen containing plant that is used as an alternative for estrogen replacement therapy. In this study, the influences of soy extract and estrogen on formalin-induced nociception in male rats were examined. In the first experiment, the effect of soy extract added to drinking water (60 mg/kg per day) on the nociception was assessed and compared with a control group (with soy-free drinking water). There was a significant decrease in nociception in rats that received soy extract for two weeks. Furthermore, different doses of soy extract (50, 150 and 200 mg/kg) were injected subcutaneously 30 min before formalin test to assess its effect on acute and chronic pain perception. It was observed that different doses of soy extract had similar antinociceptive effects that were significantly different from saline injected control group. In other experiment, rats treated with a single dose of 150 ?g of 17-? estradiol (s.c.) 48 h prior to the formalin test, to be compared with a control group just received estrogen vehicle (DMSO). Acconding to our results Estradiol did not show any significant effect on pain suppression. These results suggest that the antinociceptive effect of soy extract on chronic pain is not aresult of its phytoestrogenic activitiy, it might atherefore be related to other substances within soy extract.

In addition to its effect on reproductive organs, estrogen exhibits complex
effects on brain structure, function and behavior. In this regard, the influence
of gonadal hormones especially estrogens on nociception has recently been
accepted. Soybean is known as an important phytoestrogen containing plant that
is used as an alternative for estrogen replacement therapy. In this study, the
influences of soy extract and estrogen on formalin-induced nociception in male
rats were examined. In the first experiment, the effect of soy extract added to
drinking water (60 mg/kg per day) on the nociception was assessed and compared
with a control group (with soy-free drinking water). There was a significant
decrease in nociception in rats that received soy extract for two weeks.
Furthermore, different doses of soy extract (50, 150 and 200 mg/kg) were
injected subcutaneously 30 min before formalin test to assess its effect on
acute and chronic pain perception. It was observed that different doses of soy
extract had similar antinociceptive effects that were significantly different
from saline injected control group. In other experiment, rats treated with a
single dose of 150 μg of 17-β estradiol (s.c.) 48 h prior to the formalin test,
to be compared with a control group just received estrogen vehicle (DMSO).
Acconding to our results Estradiol did not show any significant effect on pain
suppression. These results suggest that the antinociceptive effect of soy
extract on chronic pain is not aresult of its phytoestrogenic activitiy, it
might atherefore be related to other substances within soy extract.

Keywords: Soy; Estrogen; Formalin test; Antinociception.

Introduction

There are some evidences showing sex-dependent differences between males and
females in pain perception (1). Estrogens are known as antinociceptive agents
that modulate pain sensation (2). In addition, it has been reported that during
pregnancy where there are high estrogen levels, pain threshold increases (3).
Paradoxically, it has also been reported that women are more likely to
experience more severe and longer pain than men (4). Plant-derived
phytoestrogens including soy products are molecules structurally and
functionally similar to estradiol (5, 6). There are similarities in the
structure, molecular weight and function between phytoestrogens and steroid
estrogens (7). Soy has been identified as a novel dietary ingredient that
significantly reduces hyperalgesia and neuropathic pain (8, 9). Soybean
comprises of oil, carbohydrate, fatty acids (e.g. linoleic acid and palmitic
acid), phospholipids (i.e. phosphatidyl choline, phosphatidyl ethanolamine and
phosphatidyl inositol) and minerals (calcium, iron and potassium). Isoflavones
including diadzein and genistein are from phytoestrols in soybean. Synthetic
phytoestrols resemble female hormones and have similar hormonal and non-hormonal
effects (10). So far, there has not yet been simultaneous work to compare the
phytoestrogen and estrogen influences on nociception. The aim of the present
study was to investigate the effect of soy extract administration on
formalin-induced nociception and figure out whether this effect is related to
phytoestrogenic function of soy by direct comparison with 17-β estradiol
administration in the same behavioral test.

Experimental

Experimental procedure

Adult male NMRI rats (195-220g) purchased from Razi Institue, Hesarak Iran, were
used. Every group four rats were housed in one cage at temperature 21?2?C and 12
h light-dark cycling with standard chowdier and water ad libitum. Rats were
randomly devided in nine control and experimental groups as shown in Table 1. To
determine the effect of estrogen on the nociception, rats were treated with a
single dose of 150 μg 17 β-estradiol (Sigma UK) in 100 μl of DMSO injected s.c.
48 hours prior to formalin test and compared with a control group received 100
μl of vehicle (DMSO) (10). To determine the effect of orally administrated soy
extract, animals were treated with 60 mg/kg/day soy extract added to the
drinking water for 2 weeks (11, 12) and the result was compared with a control
group used (with soy-free drinking water). The average of the consumed drinking
water was determined during one week in 10 rats and it was revealed that each
rat had approximately consumed 0.5 ml/g water per day. The drinking water with
an overestimated volume of 10% was provided daily for every group of 4 rats in
each cage. In other experimental groups rats treated with i.p. injection of soy
extract, doses of 50, 150 and 200 mg/kg 25-30 min prior to the formalin test and
were compared with saline injected control group. In positive control tests
morphine sulfate (15 mg/kg; i.p.) dissolved in saline was used (13). Animals
treated with 17β-estradiol, soy extract, morphine sulfate, DMSO or saline and
those normal feeding control rats were assessed for nociception by formalin
test.

Preparation of plant extract

The medicinal plant of soy was provided from the local market and was
scientifically identified by the department of botany of Shaheed Beheshti
University. To prepare the hydro alcoholic extract, 400 g of cleaned soy beans
was crushed and mixed at ratio of 1 to 5 with methanol 70% and kept for 48 h at
room temperature. During this time it was stirred several times and then the
deposit was separated using paper filter. After filtration with it was
maintained in a water bath at 65?C for 16 h to let the alcohol be evaported from
filtered solution to reach a final concentration of 25%.

Formalin test

Acute and chronic pain assessment was carried out via the formalin test protocol
(14). All animals were habituated to the test chamber 30 min prior to the
experiment. In all rats, 50 μl of 2.5% formalin was subcutaneously injected into
the plantar surface of hind paw. Following injection of formalin, the animals
were immediately put in a chamber with a mirror angled at 45? positioned
underneath the floor to allow an unobstructed view of the formalin injected paw
by an observer. Observations continued for the next 60 min. The first 10 min was
considered as the early phase and the period between 15 and 50-60 min as the
second phase. The pain scoring was as follows: 0, normal weight bearing on the
injected paw; 1, limping during locomotion or resting the paw lightly on the
floor; 2, elevation of the injected paw; 3, licking or biting of the injected
paw, or grooming. Behavioral responses had been reconded every 15 sec. The
average pain scores from every 3 min block in experimental groups were compared
with similar blocks in control groups. The rats were not tested more than once
and experiments were carried out between 09:00 a.m and 15:00 p.m.

Statistical analysis

Data, were expressed as means?S.E.M. Comparisons were carried out using one way
analysis of variance (ANOVA) followed by post-hoc Tukey test and p<0.05 were
considered as a significant difference.

Results

Results of administration of soy extract and estrogen are shown in Table 1.
Statistical comparison between the experimental groups (soy and estrogen) and
control groups in the first 10 min indicate that neither soy nor estrogen has
any effect on nociception in the whole first phase (early phase) of
formalin-induced pain (Figure 1 and 2). An overall comparison by statistical
analysis also indicates that oral soy administration has a significant
inhibitory effect (17% pain reduction) in the second (chronic) phase (time
interval 15-60 min) of formalin-induced nociception (P<0.001) (Figure 1 and
Table 1). However, in some periods such as 51 to 57 min, oral soy administration
did not cause any significant pain reduction. As shown in Table 1 and Figure 2,
comparing to DMSO group, estrogen (150 ?g s.c.) had no effect on pain perception
in the late phase (chronic phase) of formalin test (P>0.05).

As demonstrated in Table 1, our results showed a significant reduction of pain
perception (12% inhibition) in rats treated ci.p.injection with different doses
(50, 150, 200 mg/kg) of soy extract (P<0.001) relative to control group (treated
with saline injection). Interestingly, this result is very close to our positive
control group (morphine sulfate) that induced a 13% pain reduction (P<0.001). It
also indicates that the differences of nociception among various doses of soy
treatment are not significant. There was a similarity between oral and i.p.
treatment of soy in analgesic effect.

Discussion

The main finding of the present study is that under conditions of peripheral
inflammation such as formalin injection into the plantar surface, soy appears to
play an important role in pain relief in the second phase of formalin-evoked
nociception. However, our finding shows that oral soy consumption does not
result in alteration of formalin-induced acute phase nociception. The
suppression of heat allodynia in a chronic neuropathic pain model in another
report is in agreement with our result (9). In addition, an analgesic effect of
soy-containing diet on cancer pain model as a neuropathic pain has been reported
(15). Since there are different mechanisms for acute and chronic pain phases in
formalin test, the discrepancy in responses in two phases might be resulted from
different mechanisms. Acute pain induction is neurogenic and is mediated via the
direct effect of formalin on the nerve endings going to central nervous system,
and chronic pain is induced through peripheral pathway, resulted from
inflammatory processes (16, 17). Experimental results demonstrated that
substance P and bradykinin participate in the early phase, while histamine,
serotonin, prostaglandins and nitric oxide are involved in the late phase of the
formalin test (18). Blockade of tyrosine kinase activity and antioxidant
activity are the known effects of isoflavones (19). Other reported activities of
isoflavones, such as direct interaction with several intracellular enzymes, are
also candidate contributors to the inhibition of inflammatory response (20).
Furthermore, it is reported that genistein inhibits monoycte adhesion to
TNF-activated endothelial cells and also inhibits platelet aggregation and
release of pro-inflammatory cytokines (20, 21). Therefore, the inhibitory effect
of soy on nociceptive response in the late phase of formalin test suggests that
this effect might be due to its peripheral action and it is possibly associated
with the increases in the activities of antioxidant enzymes and also related to
the blockade of some of inflammatory mediators such as histamine, serotonin, and
prostaglandins.

It has been reported that Sabra strain rats fed with soy have reduced heat
hyperalgesia (8). However, no reduction of heat hyperalgesia (induced by
Hargreave device) in Wistar rats after nerve injury, fed with soy has been
observed (8, 12). Shir and colleagues in 2001 used Hargreave device to induce
noxious heat stimulation and acute pain wich is different from their study in
1998 (using laser) and our method in the first phase of formalin test. The acute
phase is resulted from nociceptors? activation; the chronic phase is more
reflective of inflammatory/injury-induced central and peripheral sensitization.
Hyperalgesia responses in second phase would be more comparable to nerve injury
results than acute nociceptive responses (1st phase). Therefore, the types of
noxious stimuli (formalin vs. Hargreave device or laser) or rat strain (NMRA vs.
Sabra or Wistar) are the probable reasons for different results.

Notably, it has been shown that soy-derived isoflavones such as active aglycones
(daidzein and genistein) and inactive glucosides (mainly daidzin and genistin)
did not alter pain threshold (22). Furthermore, no significant differences in
pain threshold were detected between the phytoestrogen-treated and control
groups tested with hot plate device (23). Therefore, our observation is in
agreement with these previous reports about acute pain, where the
antinociceptive effect of soy was not observed against allodynia and
hyperalgesia.

Likewise, i.p. injection of different concentrations of soy has significantly
reduced chronic pain scores in rats. The pain suppression was not significantly
different among three used concentrations. Shir and colleagues (12) have
mentioned that low and high concentrations of phytoestrogens do not induce
sensitivity reduction in neuropathic pain. Therefore, those doses used by us are
possibly in mid ranges that do not show different functions. Lower and higher
doses should be used to confirm the hypothesis. Furthermore, the non-specific
function of different doses of soy in this experiment could be another reason to
justify this subject. Since the analgesic effect of soy administration in both
oral and s.c. injection showed similarity, one can conclude that possibly the
effective ingredient of soy in nociception can pass through gastrointestinal
tract. Regarding the mechanism of second phase of formalin-induced nociception
which is related to the release of inflammatory mediators such as prostaglandin
and substance p (13), probably the antinociceptive effect of soy is mediated via
its modulatory effect on inflammatory mediators.

Our study showed that estrogen has no inhibitory effect on pain perception in
acute and chronic phases of formalin test, while we have used the same dose that
was administrated by others (10). There are paradoxical reports concerning
estrogen involvement in pain perception. For instance, it has been shown that
estrogen administration increases the proportion of heat sensitive fibers that
are also mechanosensitive (25). However, the reduction of sensitivity to noxious
stimuli during pregnancy which is accompanied by elevated gonadal hormone level
in women and female rats is also reported (26). There is evidence that the
modulatory effect of estrogen is sex-dependent, and in most cases estrogen has
excitatory and inhibitory effects on second messenger pathways in females and
males respectively. In females, estrogen can activate second messengers
including PKA, PKC and PLC and increase intracellular calcium in different
tissues such as DRG neurons via a non-genomic mechanism (27, 28). Conversely,
estrogen can inhibit PKA and PKC activation in the male rat (29, 30). It is
likely that estrogen effect on nociception is related to many parameters such as
gender, the type of pain, animal strain and other gradients that accompany
estrogen.

These information suggest that whole plant with different concentrations of
various phytoestrogens is suitable for pain relief and besides estrogen other
substances exist in soy that causes its effect. The main ingredient in soy
because of which it is involved in nociception is not known well. The most
studied candidates are phytoestrogens that inhibit protein kinase C (31).
Activation of PKC is involved in development of neuropathic pain (31). However,
phytoestrogens showed estrogenic role in some aspects of analgesic activity (8)
and further investigation with different doses of estrogen in both male and
female with measuring the estrogen plasma level could be helpful.

Conclusion

The present study showed that soy consumption (either oral or i.p. injection) in
male rats has a clear inhibitory effect on pain suppression in chronic phase.
However, neither oral administration nor i.p. injection of soy could change
nociception in early phase of formalin-induced pain. These findings suggest that
soy is possibly involved in anti-inflammatory mechanism in pain relief.
Nonetheless, estrogen did not exert any antinoceptive effect in both acute and
chronic pain in our experiment. Taken together, the hypotheses that
antinociceptive effect of soy is resulted from soy protein or estrogen or
different concentrations of isoflavones are left to be more investigated.